Source: Polymerase chain reaction
Four pahses for PCR
2n / cycle
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/PCR_Stage.jpg”, style=“zoom:20%; border:0; border-radius:20px; box-shadow:0 0 0;” alt=“PCR stage”>
Reference DOI: 10.1100/tsw.2011.124.
| Roche | Bio-Rad | Applied Biosystems | Qiagen |
|---|---|---|---|
|
LightCycler®480 <img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/LC480.jpg”, style=“zoom:30%; border:0; border-radius:20px; box-shadow:0 0 0;” alt=“LC480”> |
CFX96 TouchTM <img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/CFXconnect96.jpg”, style=“zoom:80%; border:0; border-radius:10px; box-shadow:0 0 0;” alt=“CFXConnect96”> |
StepOneTM <img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/ABIStepOne.jpeg”, style=“zoom:100%; border:0; border-radius:10px; box-shadow:0 0 0;” alt=“ABIStepOne”> |
Rotor-Gene Q |
| Gene detection | Gene expression |
|---|---|
|
|
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/qPCR_Trend.jpg” style=“zoom:30%; border:0; border-radius:20px; box-shadow:0 0 0;”, alt=“qPCR Trend”>
Number of publications in PubMed related to qPCR.
This plot shows the number of publications in PubMed related to specific qPCR applications.
Reference DOI: 10.1016/j.bdq.2014.08.002
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/qPCR_FoodAnalysis.png” style=“zoom:9.8%; border:0; border-radius:30px; box-shadow:0 0 0;”, alt=“qPCR for food analysis”>
Summary of the research articles published in different journals.
The data was retrieved from Scopus by using the key word “Real-Time PCR for Food Analysis”
Reference DOI: 10.1007/s13197-016-2205-0
Non-specific dsDNA binding dyes
Sequence-specific fluorescence detection chemistry
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/SYBR_1.jpg” style=“zoom:8%; border:0; border-radius:20px; box-shadow:0 0 0;”, alt=“SYBR green 1”>
Reference DOI: 10.1038/sj.leu.2402922
Reference DOI: 10.1002/9780470027318.a1406.pub2
Reference DOI: 10.1038/sj.leu.2402922
Reference DOI: 10.1007/s13197-016-2205-0
曾經有過這樣的設計 (142-232),看起來好像不錯!?
結果,因為立體結構的關係,使得擴增效率非常差!
Reference DOI: 10.1016/j.bdq.2014.08.002
Cp calculation
Abosolute
Relative
Tm calling analysis (Melting curve analysis)
Crossing poing (Cp) = Cycle quantification (Cq) = Cycle threshold (Ct) = Take-off point (TOP)
Picture source:What Is A Cycle Threshold (Ct) Value In qPCR?
\(N = N_{0}\times E^{Cp}\)
\(LogN = LogN_{0}+Cp\times LogE\)
\(Cp=-(\frac{1}{LogE})LogN_{0}+\frac{LogN}{LogE}\)
Reference DOI: 10.1002/9780470027318.a1406.pub2
Reference DOI: 10.5114/aoms.2014.44871
\(Relative\ ratio = \frac{Target\ gene\ concentration}{Reference\ gene\ concentration}\)
Reference gene:
Reference DOI: 10.1016/j.bbrc.2003.11.177 (被引用1,367次)
Optional:
\(Calibrator\ Normalize\ Ratio = \frac{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Sample)}{{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Calibrator)}}\)
Calibrator:
Correct the differences in detection sensitivity between Target / Reference gene and several experiments (e.g., long-term studies)
Not treated cell line
Sample before experiment
Reference: Roche 教育訓練專員講義
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/DiffMelt.jpg” style=“zoom:30%; border:0; border-radius: 10px; box-shadow:0 0 0;”, alt=“Different Melting cruve”>
Reference DOI: 10.1016/j.mcp.2010.04.002
Minimum Information for Publication of Quantitative Real-Time PCR Experiments, (2009)
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/MIQE2016.jpg” style=“zoom:20%; border:0; border-radius: 10px; box-shadow:0 0 0;”, alt=“MIQE 2016”>
<img src=“/Volumes/Data/實驗室準則/qPCR 訓練課程/qPCR_slide/Pictures/GuidesForqPCR_1.jpg” style=“zoom:20%; border:0; border-radius: 10px; box-shadow:0 0 0;”, alt=“Other guides”>
限定使用Roche原廠耗材!
(盤子與封模,唯一限定的材料)